Project Summary: Polycomb repressive complex 2 (PRC2) mediates trimethylation of histone H3 lysine 27 (H3K27me3), a hallmark for gene silencing and facultative heterochromatin formation. PRC2 consists of four core subunits, including the catalytic subunit Ezh2, Eed, Suz12, and Rbbp4, which together respond to a myriad of developmental cues. PRC2 catalysis on chromatin substrate has been known to be subjected to several layers of regulation, including chromatin context-dependent control of the enzyme activity of PRC2, auxiliary nuclear protein factor-mediated recruitment of PRC2 to chromatin, and Ezh2 phosphorylation-dependent regulation of Ezh2 activity in the canonical gene repression and the non-canonical gene activation processes. Dysregulation of PRC2 and in particular mutation of Ezh2 are broadly linked to human disease including cancer and developmental disorder. Indeed, many regulatory mechanisms of PRC2 to be studied in depth in our proposed experiments are associated with disease phenotypes. In this regard, our research may identify novel mechanism-based targets and avenues for future therapeutics. Our long-term objective is to elucidate the structural mechanisms whereby PRC2 regulates gene expression during development by controlling chromatin structure and transcriptional state. Specifically, in this research, we will address the following three key questions concerning PRC2 catalysis and regulation. (1) What are the catalytic mechanism of PRC2, including cofactor binding, substrate recognition, and allosteric regulation? (2) How is PRC2 recruited to chromatin by auxiliary nuclear protein factors, including Aebp2, Phf19, and Jarid2? (3) How are the gene repression and activation function of Ezh2 mechanistically connected and regulated, in particular by Ezh2 phosphorylation? Using a combined structural biology, biochemistry, and chemical biology approach and in particular benefiting from our established expertise and strength in structural biology, we will be focused on several PRC2-centered multi-subunit macromolecular complexes to address each of these key questions.